Measurement of Cyclin E Genomic Copy Number and Strand Length in Cell-Free DNA Distinguish Malignant versus Benign Effusions

Abstract

Purpose: Previous studies have shown that the concentration of cell-free DNA was higher and its strand length longer in body fluids obtained from patients with cancer as compared to patients with benign diseases.We hypothesized that analysis of both DNA copy number and strand length of cell-free DNA from an amplified chromosomal region could improve the diagnosis of malignant diseases in body fluids. Experimental Design: To test this hypothesis, we used ovarian cancer effusion as an example and applied a quantitative real-time PCR to measure the relative copy number and strand length of DNA fragments from one of the most frequently amplified genes, cyclin E, in ovarian serous carcinomas. Results: As compared with nonamplified chromosomal loci, including h-actin, p53, 2p24.1, and 4p15.31, measurement of cyclin E DNA copy number (100 bp) had the best performance in distinguishing malignant (n = 88) from benign (n = 70) effusions after normalization to effusion volume or Line-1 DNA with areas under the receiver operating characteristics curve (AUC) of 0.832 and 0.847, respectively. Different DNA lengths of the cyclin E locus were further analyzed and we found that the AUC was highest by measuring the 400-bp cyclin E locus (AUC = 0.896). The AUC was improved to 0.936 when it was combined with the length integrity index as defined by the relative abundance of 400 bp cyclin E to 100 bp p53 loci. Cyclin E real-time PCR assay had a higher sensitivity (95.6%) than routine cytology examination (73.9%) and was able to diagnose false-negative cytology cases in this study. Conclusions: The above findings indicate that measurement of the DNA copy number and strand length of the cyclin E locus is a useful cancer diagnostic tool.

Pre-analytical issues in effusion cytology

“…Another diagnostic approach utilizing effusion supernatants is through analysis of secreted DNA fragments, as shown for CCNE1, encoding cyclin E. Archival material was used in this study too [43]. Analysis of the mRNA levels of BIRC5, encoding the anti-apoptotic marker Survivin, by qPCR, showed overexpression of this mRNA in supernatants from malignant compared to benign effusions [44].…”

Cell-free DNA as a diagnostic marker for cancer: current insights

“…The authors also studied the integrity index of cyclin E. Combination of cyclin E copy number (400 bp) and DNA length index had an AUC value of 0.936 34. Since the analysis of cyclin E had a higher sensitivity (95.6%) than cytology (74%), it may be suitable for false-negative cytology cases.…”

Monitoring tumor-derived cell-free DNA in patients with solid tumors: Clinical perspectives and research opportunities

“…In ovarian cancer patients were investigated also the MSI and the DNA integrity with promising results [97] and [98].…”